RESUMEN
Reporter genes produce a protein product in transfected cells that can be easily measured in intact or lysed cells and they have been extensively used in numerous basic and applied research applications. Over the past 10 years, reporter gene assays have been widely accepted and used for analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related dioxin-like compounds in various types of matrices, such as biological, environmental, food and feed samples, given that high-resolution instrumental analysis techniques are impractical for large-scale screening analysis. The most sensitive cell-based reporter gene bioassay systems developed are the mechanism-based CALUX (Chemically Activated Luciferase Expression) and CAFLUX (Chemically Activated Fluorescent Expression) bioassays, which utilize recombinant cell lines containing stably transfected dioxin (AhR)-responsive firefly luciferase or enhanced green fluorescent protein (EGFP) reporter genes, respectively. While the current CALUX and CAFLUX bioassays are very sensitive, increasing their lower limit of sensitivity, magnitude of response and dynamic range for chemical detection would significantly increase their utility, particularly for those samples that contain low levels of dioxin-like HAHs (i.e., serum). In this study, we report that the addition of modulators of cell signaling pathways or modification of cell culture conditions results in significant improvement in the magnitude and overall responsiveness of the existing CALUX and CAFLUX cell bioassays.
RESUMEN
Chronic beryllium disease (CBD), an irreversible, debilitating granulomatous lung disease is caused by exposure to beryllium. This occupational hazard occurs in primary production and machining of Be-metal, BeO, beryllium - containing alloys, and other beryllium products. CBD begins as an MHC Class II-restricted, T(H)1 hypersensitivity, and the Human Leukocyte Antigen, HLA-DPB1E(69), is associated with risk of developing CBD. Because inbred strains of mice have not provided good models of CBD to date, three strains of HLA-DPB1 transgenic mice in an FVB/N background were developed; each contains a single allele of HLA-DPB1 that confers a different magnitude of risk for chronic beryllium disease: HLA-DPB1*0401 (OR approximately 0.2), HLA-DPB1*0201 (OR approximately 3), and HLA-DPB1*1701 (OR approximately 46). The mouse ear swelling test (MEST) was employed to determine if these different alleles would support a hypersensitivity response to beryllium. Mice were first sensitized on the back and subsequently challenged on the ear. In separate experiments, mice were placed into one of three groups (sensitization/challenge): C/C, C/Be, and Be/Be. In the HLA-DPB1*1701 mice, the strain with the highest risk transgene, the Be/Be group was the only group that displayed significant maximum increased ear thickness of 19.6% +/- 3.0% over the baseline measurement (p < 0.05). No significant changes were observed in the other transgenic strains for any treatment condition. In addition, inter-strain differences in response to beryllium in seven inbred strains were investigated through use of the MEST, these included: FVB/N, AKR, Balb/c, C3H/HeJ, C57/BL6, DBA/2, and SJL/J. The FVB/N strain was least responsive, while the SJL/J and C57/BL6 strains were the highest responders. Our results suggest that the HLA-DPB1*1701 transgene product is an important risk factor for induction of the beryllium-sensitive phenotype. This model should be a useful tool for investigating beryllium sensitization.
Asunto(s)
Beriliosis/genética , Beriliosis/inmunología , Modelos Animales de Enfermedad , Antígenos HLA-DP/genética , Hipersensibilidad Tardía/genética , Hipersensibilidad Tardía/inmunología , Alelos , Animales , Berilio/efectos adversos , Predisposición Genética a la Enfermedad , Antígenos HLA-DP/metabolismo , Cadenas beta de HLA-DP , Humanos , Hipersensibilidad Tardía/inducido químicamente , Ratones , Ratones Endogámicos , Ratones Transgénicos , Polimorfismo Genético , Factores de Riesgo , Pruebas Cutáneas , Especificidad de la Especie , Células TH1/inmunologíaRESUMEN
Expression of Cyp1a1 and its related enzyme activity have long been used as a biomarker for aryl hydrocarbon receptor (AhR) activation and a warning of dioxin-like toxicity. As a result, induction of Cyp1a1 by pharmaceutical drug candidates or environmental contaminants raises significant concern in risk assessment. The current study evaluates the specificity of Cyp1a1 induction as a marker for AhR affinity and activation and provides context to assess the relevancy of AhR activation to risk assessment. In vivo experiments examined the expression of Cyp1a1 and other AhR-regulated genes in liver, kidney, and heart in response to 596 compounds. From this data set, a subset of 147 compounds was then evaluated for their ability to activate or bind to the AhR using a combination of gel shift, reporter gene, and competitive receptor binding assays. Whereas in vivo Cyp1a1 mRNA expression is a sensitive marker for AhR activation, it lacks specificity, because 81 (59%) of 137 compounds were found to significantly induce Cyp1a1 in vivo but were not verified to bind or activate the AhR in vitro. Combining in vivo and in vitro findings, we identified nine AhR agonists, six of which are marketed therapeutics and have been approved by the U.S. Food and Drug Administration, including leflunomide, flutamide, and nimodipine. These drugs do not produce dioxin-like toxicity in rats or in humans. These data demonstrate that induction of Cyp1a1 is a nonspecific biomarker of direct AhR affinity and activation and lend further support to the hypothesis that Cyp1a1 induction and/or AhR activation is not synonymous with dioxin-like toxicity.